Metabolic engineering, encompassing application of recombinant DNA technology, has shown its potential to optimize cellular functions for many purposes: recombinant protein production, pathway engineering for productivity enhancement, and novel pathway design for new product generation. Defined as a sequence of conversions that is not found in host species, a novel pathway has been designed and constructed in E. coli for the production of 1,3-propanediol (C. E. Nakamura and G. M. Whited (2003). Curr. Opin. Biotechnol. 14: 454-459), amorphadiene (Nature Biotech, 21, pp 796-802), and 1,2,4-butanetriol (JACS, 125, pp 12998-12999). In these approaches, each step was designed based on enzyme availability, the recruited enzyme activities from various organisms were identified, and the novel pathways were constructed in E. coli by assembling these enzymatic steps. The basic idea behind these examples is to consider proteins including enzymes as interchangeable parts, and the term “synthetic biology” has been used to describe this concept (Nature 421, p 118; Nature Chemical Biology, 3, pp 521-525).
D-glucaric acid is found in fruits, vegetables, and mammals and has been studied for cholesterol reduction (Z. Walaszek, et al. (1996). Nutr. Res. 16: 673-681) and cancer chemotherapy (J. Singh and K. P. Gupta (2003). Biomed. Environ. Sci. 16: 9-16). In a recent report (T. Werpy and G. Petersen (2004). “Top Value Added Chemicals From Biomass,” Vol. I, PNNL and NREL), D-glucaric acid was identified as a “Top Value Added Chemicals From Biomass” and as a promising starting material for producing new nylons and hyperbranched polyesters. D-glucaric acid, a highly functionalized compound with four chiral carbons, is currently produced by chemical oxidation of D-glucose, a nonselective and expensive process using nitric acid as the oxidant (T. Werpy and G. Petersen (2004). “Top Value Added Chemicals From Biomass,” Vol. I, PNNL and NREL). New catalytic processes using enzymes may lead to higher yield and selectivity. The biological approach for producing glucaric acid could be made by mimicking the existing D-glucuronic acid pathway in mammals. However, this is an inefficient pathway, which consists of more than ten conversion steps, starting with D-glucose.